Oligodendrocytes (OLs) are the end product of a well-characterized developmental lineage in which the cells pass through distinct phenotypic stages under the control of both intrinsic and extrinsic factors. This process culminates in a precise reorganization of morphology and the production of dramatic amounts of myelin in a unique association with neurons, leading to saltatory conduction and dramatic savings in energy and space. Myelin is a dynamic, functionally active membrane whose loss or damage results in serious neurological deficits such as occur in Multiple Sclerosis. AIM I. The decision to cease proliferation and initiate terminal differentiation is a critical stage in cellular differentiation. We investigated a MODEL that proposes that glycosphingolipids, in particular galactocerebroside (GalC) and/or sulfatide, are involved in negative regulation of OL differentiation. A key finding is that antibodies against GalC/sulfatide, block entry of OLs into terminal differentiation. This block is non-toxic, reversible and operates at the level of mRNA; thus, it offers a unique tool for studying the mechanism of regulation of entry into terminal differentiation. In contrast, and consistent with the model, in GalC/sulfatide null mice, terminal differentiation is enhanced. The proposed participation in this regulation of cell adhesion molecules, src-family kinases, and FGF receptor-3 will be investigated. AIM II. As OLs enter terminal differentiation, they begin to produce myelin membrane on a remarkable scale and initiate contact with neuronal axons. We investigate the mechanism by which OLs sort and transport myelin lipids and proteins from the trans-Golgi network to the neo-myelin membrane to produce a highly polarized cell with multiple submembrane domains. A combination of immuno-isolation and immuno-imaging will be used to study the formation of discrete populations of transport vesicles and their regulated passage, docking and fusion with presumed specific sites in the plasma membrane. The roles of tethering complexes thought to target transport vesicles to unique sites of membrane insertion will be investigated, beginning with an analysis of the sec6/8 complex. Important experimental tools include a highly characterized primary OL cell culture system; 2D-PAGE/tandem mass spectrometric proteomic analysis; analysis of glycosphingolipid signaling microdomains; mutant mice null for GalC and/or sulfatide or FGFR3; high resolution confocal imaging and fluorescently-tagged myelin and trafficking proteins for real-time image analysis; genetic modification of primary OLs for functional analyses. A long-term goal of this project is to develop a detailed understanding of the molecular mechanisms of OL differentiation and myelin biogenesis in order to contribute to an informed intervention in clinical settings that will encourage more substantial remyelination in demyelinating diseases